1. Field of the Invention
The present invention relates generally to the field of molecular biology, stem cells and differentiated cells. More particularly, it concerns maturation of pluripotent stem cell-derived hepatic lineage cells that are receptive to virus infection, particularly hepatitis B virus.
2. Description of Related Art
In vitro model systems based on hepatocytes have been used to better understand the role of hepatocytes in physiological processes of the liver and in studying diseases or conditions of the liver such as hepatitis B infection (HBV), cirrhosis, liver injury, and hepatocellular carcinoma. These model systems are also used for drug metabolism studies, screening of drugs such as anti-HBV drugs, and cell-based therapy for the treatment of liver diseases. While primary human hepatocytes have been commonly used in biological and biopharmaceutical research, they are in limited supply as they do not proliferate in culture. Moreover, primary human hepatocytes rapidly lose their hepatic phenotype shortly after isolation from the in vivo environment (Guillouzo et al., 1998), and the drug metabolic ability of human primary hepatocytes exhibits significant differences between donors. In addition, while primary human hepatocytes support HBV infection, the infection is not robust even with supplementation of cell-culture medium with dimethyl sulfoxide or polyethylene glycol (Gripon et al., 1988; Gripon et al., 1993).
The ability to direct the differentiation of human pluripotent stem cells (PSCs) such as induced pluripotent stem cells and embryonic stem cells to specific lineages including hepatocytes provides access to unlimited numbers of human hepatocytes for a wide range of applications that include development of new treatments for a spectrum of diseases, the establishment of platforms for drug discovery and predictive toxicology and the creation of in vitro models of disease. The availability of an unlimited supply of patient-specific functional hepatocytes would greatly facilitate both the drug development and the eventual clinical application of hepatocyte transplantation. Thus, there has been significant effort to develop PSC-derived hepatocytes that recapitulate the properties of in situ primary hepatocytes as an alternative, unlimited source of hepatocytes. In general, these methods induce differentiation of a monolayer of PSCs with the addition of pathway agonists and antagonists that are known to regulate endoderm induction and hepatic specification. For example, hepatocyte growth factor (HGF) was shown to synergize with activin A and Wnt3a for endodermal induction and subsequent culture in maturation media containing oncostatin M resulted in hepatocytes positive for albumin (Chen et al., 2012).
However, the currently known protocols for producing PSC-derived hepatocytes produce immature hepatocytes that do not display functional levels of key drug-metabolizing enzymes and do not efficiently replicate hepatitis B virus. Therefore, there is a need for methods to produce more mature PSC-derived hepatocytes for therapeutic and research use, especially hepatocytes capable of hepatitis B virus replication for use as models in screening for anti-HBV drugs.